Special NOESY experiments Filtered,edited NOE:based H Labeled on selection of NOEs from two Unlabeled protein peptide 13C molecules with unique labeling patterns. Only NOEs at the interface .Transferred NOE:based on 1).faster build-up of NOEs in large versus small molecules;2) Fast exchange 3)NOEs of koff bound state detected at resonance frequencies of free state Only NOEs from bound state 16
16 Special NOESY experiments •Filtered, edited NOE: based on selection of NOEs from two molecules with unique labeling patterns. Unlabeled peptide Labeled protein Only NOEs at the interface Only NOEs from bound state H H H H kon koff •Transferred NOE: based on 1) faster build-up of NOEs in large versus small molecules; 2) Fast exchange 3) NOEs of bound state detected at resonance frequencies of free state
H-1H distances from NOEs Long-range (tertiary structure) Sequential ee心告…古 Medium-range (helices) Challenge is to assign all peaks in NOESY spectra 17
17 1H- 1H distances from NOEs Intra-residue A B C D • • • • Z Sequential Medium-range (helices) Long-range (tertiary structure) Challenge is to assign all peaks in NOESY spectra
NMR data 1:NOE Conversion of NOE into distances -Strong: 1.8-2.7 -Medium: 1.8-3.3A -Weak: 1.8-5 Lower bound because of vdw radii of atoms 18
NMR data 1: NOE • Conversion of NOE into distances – Strong: 1.8 - 2.7 Å – Medium: 1.8 - 3.3 Å – Weak: 1.8 - 5 Å Lower bound because of vdw radii of atoms 18
NOE pseudo-energy potential Generate "fake"energy potentials representing the cost of violating the distance or angle restraints.Here's an example of a distance restraint potential KNoE(r)2 ifryr ifry<r<n KNoE(rru)2 if ry<r where rand r"are the lower and upper bounds of our distance restraint, and KNo is some chosen force constant,typically ~250 kcal mol-1 nm2 So it's somewhat permissible to violate restraints but it raises V 19
NOE pseudo-energy potential • Generate “fake” energy potentials representing the cost of violating the distance or angle restraints. Here’s an example of a distance restraint potential KNOE(rij-rij 1 ) 2 if rij<rij l KNOE(rij-rij u ) 2 if rij>rij u 0 if rij l<rij < rij u VNOE = where rij l and rij u are the lower and upper bounds of our distance restraint, and KNOE is some chosen force constant, typically ~ 250 kcal mol-1 nm-2 So it’s somewhat permissible to violate restraints but it raises V 19
NOE pseudo-energy potential VNOE Potential rises steeply with degree of violation 20
NOE pseudo-energy potential rij l rij u 0 VNOE Potential rises steeply with degree of violation 20